Note: Descriptions are shown in the official language in which they were submitted.
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FURAN NITRONE THERAPEUTICS
FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
The application claims the benefit of U.S. Patent Application Serial No.
60/0$5,974, filed May 19, 1998, the disclosure of which is incorporated herein
by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to the treatment of inflammatory bowel disease
(IBD). More specifically, this invention is directed to methods for treating
or
preventing IBD using furan nitrone compounds. This invention is also directed
to pharmaceutical compositions containing furan nitrone compounds which are
useful for the treatment or prophylaxis of IBD.
The term inflammatory bowel disease ("IBD") describes a group of
chronic inflammatory disorders of unknown causes involving the
gastrointestinal
tract ("GI tract"). The prevalence of IBD in the US is estimated to be about
200
per 100,000 population or approximately 500,000 people. Patients with IBD can
be divided into two major groups, those with ulcerative colitis ("UC") and
those
with Crohn's disease ("CD")
In patients with UC, there is an inflammatory reaction primarily
involving the colonic mucosa. The inflammation is typically uniform and
continuous with no intervening areas of normal mucosa. Surface mucosal cells
as well as crypt epithelium and submucosa are involved in an inflammatory
reaction with neutrophil infiltration. Ultimately, this situation typically
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progresses to epithelial damage with loss of epithelial cells resulting in
multiple
ulcerations, fibrosis, dysplasia and longitudinal retraction of the colon.
CD differs from UC in that the inflammation extends through all layers of
the intestinal wall and involves mesentery as well as lymph nodes. CD may
affect any part of the alimentary canal from mouth to anus. The disease is
often
discontinuous, i.e., severely diseased segments of bowel are separated from
apparently disease-free areas. In CD, the bowel wall also thickens which can
lead to obstructions. In addition, fistulas and fissures are not uncommon.
Clinically, IBD is characterized by diverse manifestations often resulting
in a chronic, unpredictable course. Bloody diarrhea and abdominal pain are
often accompanied by fever and weight loss. Anemia is not uncommon, as is
severe fatigue. Joint manifestations ranging from arthralgia to acute
arthritis as
well as abnormalities in liver function are commonly associated with IBD.
Patients with IBD also have an increased risk of colon carcinomas compared to
the general population. During acute "attacks" of IBD, work and other normal
activity are usually impossible, and often a patient is hospitalized.
Although the cause of IBD remains unknown, several factors such as
genetic, infectious and immunologic susceptibility have been implicated. IBD
is
much more common in Caucasians, especially those of Jewish descent. The
chronic inflammatory nature of the condition has prompted an intense search
for
a possible infectious cause. Although agents have been found which stimulate
acute inflammation, none has been found to cause the chronic inflammation
associated with IBD. The hypothesis that IBD is an autoimmune disease is
supported by the previously mentioned extraintestinal manifestation of IBD as
joint arthritis, and the known positive response to IBD by treatment with
therapeutic agents such as adrenal glucocorticoids, cyclosporine and
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azathioprine, which are known to suppress immune response. In addition, the GI
tract, more than any other organ of the body, is continuously exposed to
potential
antigenic substances such as proteins from food, bacterial byproducts (LPS),
etc.
Once the diagnosis has been made, typically by endoscopy, the goals of
therapy are to induce and maintain a remission. The least toxic agents which
patients are typically treated with are the aminosalicylates. Sulfasalazine
{Azulfidine), typically administered four times a day, consists of an active
molecule of aminosalicylate (5-ASA) which is linked by an azo bond to a
sulfapyridine. Anaerobic bacteria in the colon split the azo bond to release
active
5-ASA. However, at least 20% of patients cannot tolerate sulfapyridine because
it is associated with significant side-effects such as reversible sperm
abnormalities, dyspepsia or allergic reactions to the sulpha component. These
side effects are reduced in patients taking olsalazine. However, neither
sulfasalazine nor olsalazine are effective for the treatment of small bowel
inflammation. Other formulations of 5-ASA have been developed which are
released in the small intestine (e.g. mesalamine and asacol). Normally it
takes 6-
8 weeks for 5-ASA therapy to show foil efficacy.
Patients who do not respond to 5-ASA therapy, or who have a more
severe disease, are prescribed corticosteroids. However, this is a short term
therapy and cannot be used as a maintenance therapy. Clinical remission is
achieved with corticosteroids within 2-4 weeks, however the side effects are
significant and include a Coshing goldface, facial hair, severe mood swings
and
sleeplessness. The response to sulfasalazine and 5-aminosalicylate
preparations
is poor in Crohn's disease, fair to mild in early ulcerative colitis and poor
in
severe ulcerative colitis. If these agents fail, powerful immunosuppressive
agents such as cyclosporine, prednisone, 6-mercaptopurine or azathioprine
(converted in the liver to 6-mercaptopurine) are typically tried. For Crohn's
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disease patients, the use of corticosteroids and other immunosuppressives must
be carefully monitored because of the high risk of intra-abdominal sepsis
originating in the fistulas and abscesses common in this disease.
Approximately
25 % of IBD patients will require surgery (colectomy) during the course of the
disease.
Oxygen-derived free radicals such as HO ~ , the superoxide anion and
other reactive oxygen species such as HOCI, have emerged as a common
pathway of tissue injury in a wide variety of diseases whose underlying cause
is
an inappropriately vigorous and sustained immune response (failure to control
or
down regulate response to the initial, appropriate stimulus). Examples of
other
diseases, in addition to IBD and arthritis, where this mechanism appear to be
the
operative cause are ARDS, septic shock, asthma, diabetes, multiple sclerosis,
uveitis, etc. Typically, both a cytokine-mediated immune response and a
nonspecific inflammatory cascade are involved in the primary inappropriate
response with both responses mediated through active oxygen species {oxidative
stress). The inappropriate secondary response, also mediated through oxidative
stress) may involve tissue damaging oxidation by neutrophils and tissue
macrophages.
Various approaches have been taken to suppress this inappropriate
inflammatory response. Small molecule inhibitors of the various leukotriene,
PAF and cyclooxygenase pathways have shown only limited efficacy, perhaps
because blocking only one of many pathways does not provide a sufficiently
large decrease in overall oxidative stress. Another approach has been the use
of
antibodies or cloned receptor molecules which target specific proteins in the
inflammatory cascade such as IL-1, IL-6 or TNF-a. However, this approach is
practical only for acute conditions, like septic shock or ARDS, where IV
administration and antibody formation against the therapeutic protein is less
of a
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concern. For a chronic condition like IBD, an orally active small molecule
that
is fully active when dosed once-a-day would be the preferred method of
treatment.
Another approach to mitigating the oxidative stress resulting from an
inflammatory response is to employ nitrone-related therapeutics (NRTs). The
prototype NRT is a-phenyl-t-butyl nitrone (PBN) shown below.
O_
/ i N+ CH3
~CH3
H CH3
NRTs represent a new category of therapeutics with the inherent capacity
to overcome the shortcomings of other previously studied compounds. Among
other properties, NRTs such as PBN are believed to trap free radicals (R~) by
adding the radical to form a more unreactive nitroxyl free radical.
Nitrones were first used as analytical tools capable of reacting with highly
reactive radicals to yield free radical adducts that are much less reactive.
In
many cases, the free radical/nitrone adduct complex is stable enough to allow
in
vivo isolation and quantitation using electron spin resonance (ESR). The
concept
of using nitrones as therapeutics in, for example, neurodegenerative diseases
resulted from the observations that nitrones, such as PBN, trap reactive
oxygen
species and/or secondary free radicals following ischemia. The therapeutic
effects of nitrones may result because the nitrones convert highly reactive
radicals into much less reactive products. Certain NRTs have been shown to
protect experimental animals from ischemia/reperfusion injury (stroke). NRTs,
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administered chronically, reverse the age-associated increase in oxidatively
damaged protein and the age-associated decrease in the activity of the
oxidative-
sensitive enzyme, glutamine synthetase, in the brain.
Accompanying the NRT-mediated changes in oxidized protein and
glutamine synthetase activity is a significant improvement in the performance
of
animals in behavioral tests measuring short-term spatial memory. For example,
it has been shown that prototype NRTs mitigate the effects of this
inflammatory
cascade in a number of in vivo models. Of particular interest is the
consistent
and well documented protection shown by PBN against the lethality induced by
LPS in various rodent models of septic shock. Remarkably, PBN has also been
shown to increase the life span of senescence-accelerated mice by one third,
perhaps by mitigating free radical damage. PBN has also been shown to block
inducible nitric oxide synthetase ("iNOS"), the enzyme responsible for
producing
large amounts of the highly damaging NO~. Thus, PBN can both trap HO~ and
suppress formation of NO~, potentially neutralizing the effects of the two
agents
considered to be the most damaging to tissue.
When evaluating the prospects of using an antioxidant to successfully
treat IBD, it is perhaps also useful to consider that the anti-oxidant defense
of the
human colon is relatively deficient compared to human liver (mucosal levels of
SOD, catalase and GSH representing 8 % , 4 % and 40 % , respectively of liver
levels), thus leaving the colon particularly sensitive to oxidative stress.
A considerable number of chemical modifications have been made to increase
NRTs suitability as therapeutic agents. The effects of intrinsic chemical
reactivity and radical trapping ability have been examined by substituting the
phenyl ring with electron donating or electron withdrawing substituents. More
water soluble analogues have also been made which, for example, have a
carboxylate or sodium sulfonate group on the phenyl ring. In addition,
lipophilic
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analogues have been made with functional group substitutions on either the
phenyl ring or the nitronyl nitrogen. The alkyl nitrogen substituent has also
been
varied through the standard straight chain and branched C3-CS substituents.
Nitrone isosteres and related compounds have also targeted and examined for
efficacy. This approach has led to various classes of compounds, such as
substituted ureas, amides, thioamides, azoxy derivatives, sulphones, and
hydroxamic acids. Among these, some benzamide compounds substantially
similar in structure to some nitrones, such as PBN, have been shown to have
activity in the treatment of Parkinson's disease, HIV dementia, and related
conditions .
As a final aspect of background, in evaluating the effectiveness of
compounds in the treatment of IBD, an in vivo model based upon trinitrobenzene
sulfonic acid ("TNBS") is used.
References relating to the above-mentioned subjects include:
Gtickman, RM (1994) Inflammatory Bowel Disease in ~j.~rr's~on'_s
Principles of Internal Medicine (McGraw Hill, New York, NY) Chapter
255: 1403-1416.
Catkins, BM, Mendetoff, A1 (1986) Epidemiology of Inflammatory
Bowel Disease, Epidemiology Review 8: 60-90.
Levin, B. (1992) Inflammatory Bowel Disease and Colon Cancer, Cancer
(Supplement), 70: 1313-1316.
Crotty. B. (1994) Ulcerative Colitis and Xenobiotic Metabolism, Lancet,
343: 35-38.
Hanauer, SB, Baert, F. (1994) Medical Therapy of Inflammatory Bowel
Disease, Med Clin North Am, 78: 1413-1426.
MacDermott, RP (1994) Alterations in the Mucosal System in Ulcerative
Colitis and Crohn's Disease, Med Clin North Am, 78: 1207-1231.
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Hanauer, B. (1993) Medical Therapy of Ulcerative Colitis, Lancet, 342:
412-417.
Winnow, VR, Winyard, PG, Morris, CJ, Blake, DR (1993) Free radicals
in Inflammation: Second Messengers and Mediators of Tissue
Destruction, Br Med Bull 49: 506-522.
Floyd, RA and Carney, J., Nitrone Radical Traps (NRTs) Protect in
Experimental Neurodegenerative Diseases, in ~leuroprotective
~,gproaches 1~, the Treatment of Parkincnn's i~isease and Other
Neurodeg~enerative Disorders (Olanow, CW, Jenner, P and Youssim E,
Eds.) Academic Press, New York, New York, in press.
Cao, X. and Phillis, JW (1994) a-Phenyl-N-tent-butyl-nitrone Reduces
Cortical Infarct and Edema in Rats Subjected to Focal Ischemia. Brain
Res. 644: 267-272.
Zhao, Q., Pahlmark, K., Smith, M.-J., and Siesjo, B. (1994) Delayed
Treatment with the Spin Trap a-phenyl-n-tent-butyl nitrone (PBN)
Reduces Infarct Size Following Transient Middle Cerebral Artery
Occlusion in Rats. Acta Physiol. Scad. 152: 349-350.
Oliver, CN, Starke-Reed, PE, Stadtman, ER, Carney, JM and Floyd, RA
(1990) Oxidative Damage to Brain Proteins, Los of Glutamine Synthetase
Activity and Production of Free Radicals During Ischemia Induced Injury
to Gerbil Brain. Proc. Natl. Acad. Sci. USA 87: 5144-5147.
Carney, JM, Starke-Reed, PE Oliver, CN, Landrum, RW, Cheng, MS,
Wu, JF and Floyd, RA (1991) Reversal or age-related increase in brain
protein oxidation in enzyme activity, and loss in temporal and spatial
memory by chronic administration of the spin-trapping compound N-tert-
butyl-a-phenylnitrone. Proc. Natl. Acad. Sci., 88: 3633-3636.
Novelli, GP (1992) Oxygen Radicals in Experimental Shock: Effects of
Spin-Trapping Nitrones in Ameliorating Shock Pathophysiology, Critical
Care Medicine, 20: 499-507.
Hamburger, SA, McCay, PB (1989) Endotoxin-Induced Mortality in Rats
is Reduced by Nitrones, Circulatory Shock, 29: 329-334.
Progrebniak, HW, Merino, MJ, Hahn, SM, Mitchell, JB, Pass, HI
( 1992) Spin Trap Salvage from Endotoxemia: The Role of Cytokine
Down-Regulation, Surgery, 112: 130-139.
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McKechnie, K., Furman, BL, Paratt JR (1986), Modification by Oxygen
Free Radical Scavengers of the Metabolic and Cardiovascular Effects of
Endotoxin Infusion in Conscious Rats, Circulatory Shock 19: 429-439.
Edamatsu,R, Mori,A., Packer, L (1995) The Spin Trap N-tert-a-phenyl-
butylnitrone Prolongs the Life Span of the Senescence Accelerated
Mouse, Biochem Biophys Res Comm 211: 847-849.
Miyajima, T., Kotake, Y. (1995) Spin Trapping Agent, Phenyl N-
Tert_Butyl Nitrone, Inhibits Induction of Nitric Oxide Synthase in
Endotoxin-Induced Shock in Mice, Biochem Biophys Res Commun, 215:
114-121.
Boettner, GR (1987) ESR Parameters of Spin Adducts, Free Radical
Biology, 3: 259-303.
Harris, ML, Schiller, HJ, Reilly, PM, Donowitz, M, Grisham, MB,
Bulkley (1992), Free Radicals and Other Reactive Oxygen Metabolites in
Imflammatory Bowel Disease: Cause, Consequence or Epiphenomenom,
Pharmacol. Ther., 53: 375-408.
Grisham MB, MacDermott, RP, Deitch EA (1990), Oxidant Defence
Mechanisms in the Human Colon, Inflammation, 14: 669-680.
Elson, CO, Startor, RB, Tennyson, GS, Ridell, RH (1995), Experimental
Models of Inflammatory Bowel Disease, Gastroenterology, 109: 1344-
1367.
Yamada, T, Marshall, S, Specian, RD, Grisham, MB (1992) A
Comparative Analysis of Two Models of Colitis in Rats,
Gastroenterology, 102: 1524-1534.
Wallace, JA, MacNaughton, WK, Morris, GP, Beck PL (1989) Inhibition
of Leulotriene Synthesis Markedly Accelerates Healing in a Rat Model of
Inflammatory Bowel Disease, Gastroenterology, 95: 29-35.
Higa, A. McKnight, GW, Wallace, JL (1993) Attenuation of Epithelial
Injury in Acute Experimental Colitis by Immunomodulators, Eur. J.
Pharmacol. 239: 171-178.
Castro, GA, Roy, SA, Stockstill, RD (1974) Trichinella Spiralis:
Peroxidase Activity in Isolated Cells from the Rat Intestine, Exp.
Parasitol. , 36: 307-315 .
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SUMMARY OF THE INVENTION
It has now been found that certain furan nitrone compounds are effective
for the treatment and prophylaxis of IBD.
Accordingly, in one of its composition aspects, this invention provides a
pharmaceutical composition for the treatment or prophylaxis of inflammatory
bowel disease comprising a pharmaceutically acceptable carrier and an
effective
inflammatory bowel disease-treating amount of a compound of formula I:
O
YO-O / \ ~ N~ ~ I
O ~O~ H R
wherein
R' is selected from the group consisting of alkyl of from 4 to 12 carbon
atoms, and cycloalkyl of from 3 to 10 carbon atoms;
Y is hydrogen or a pharmaceutically acceptable cation;
and pharmaceutically acceptable salts thereof.
Preferably, R' is an alkyl group having from 3 to 8 carbon atoms, or a
cycloalkyl group having from 6 to 10 carbon atoms. More preferably, R~
selected from the group consisting of n-butyl, tent-butyl, n-hexyl,
cyclohexyl,
and adamantyl.
Preferably, Y is hydrogen ar a sodium cation.
In another of its composition aspects, this invention provides a
pharmaceutical composition for the treatment or prophylaxis of inflammatory
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bowel disease comprising a pharmaceutically acceptable carrier and an
effective
inflammatory bowel disease-treating amount of a compound selected from the
group consisting of:
N n-butyl-a-(2-sulfofuran-5-yl)nitrone,
N tent-butyl-a-(2-sulfofuran-5-yl)nitrone,
N n-hexyl-a-(2-sulfofuran-5-yl)nitrone,
N cyclohexyl-a-(2-sulfofuran-S-yl)nitrone,
N adamantyl-a-(2-sulfofuran-5-yl)nitrone
and pharmaceutically acceptable salts thereof.
Another aspect of this invention is directed to methods for treating a
patient suffering from or susceptible to an inflammatory bowel condition.
Accordingly, this invention provides a method for treating a patient suffering
from or susceptible to an inflammatory bowel condition comprising
administering to said patient a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and an effective inflammatory bowel
condition-treating amount of a compound of formula I:
O
,N
Yo
II O
O H
wherein
Rl is selected from the group consisting of alkyl of from 4 to 12 carbon
atoms, and cycloalkyl of from 3 to 10 carbon atoms;
Y is hydrogen or a pharmaceutically acceptable cation;
and pharmaceutically acceptable salts thereof.
Preferably, R' and Y are as described above.
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In another of its method aspects, this invention provides a method for
treating or preventing inflammatory bowel disease comprising:
(a) identifying a patient suffering from or susceptible to an
inflammatory bowel condition; and
(b) administering to said patient a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective inflammatory
bowel condition-treating amount of a compound of formula I above.
In still another of its method aspects, this invention provides a method for
treating a patient suffering from or suscelitible to an inflammatory bowel
condition comprising administering to said patient a pharmaceutical
composition
comprising a pharmaceutically acceptable carrier and an effective inflammatory
bowel condition-treating amount of a compound selected from the group
consisting of:
N n-butyl-a-(2-sulfofuran-5-yl)nitrone,
N tent-butyl-a-(2-sulfofuran-5-yl)nitrone,
N n-hexyl-a-(2-sulfofuran-5-yl)nitrone,
N cyclohexyl-a-(2-sulfofuran-5-yl)nitrone,
N adamantyl-a-(2-sulfofuran-5-yl)nitrone
and pharmaceutically acceptable salts thereof.
In the methods of this invention, the pharmaceutical compositions may be
administered orally, parenterally, or rectally. The methods of this invention
are
be effective where the inflammatory bowel condition is ulcerative colitis or .
Crohn's disease.
In one embodiment of the above methods, the pharmaceutical composition
is preferably administered as an oral dose in an amount of from 0.1 to about
150
mg/kg of patient weight.
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In another embodiment of the above methods, the pharmaceutical
composition is preferably administered intravenously in an amount of from
about
0.01 mg/kg/hour to about 100 mg/kg/hour of patient weight for at least about 1
hour.
In still another embodiment of the above methods, the pharmaceutical
composition is preferably administered rectally in an amount of from 1 to
about
150 mg/kg of patient weight.
In one of its composition aspects, this invention is also directed to novel
amide compounds. Accordingly, this invention is directed to the following
compound:
N adamantyl-a-(2-sulfofuran-5-yl)nitrone.
DETAILED DESCRIPTION OF THE INVENTION
The treatment methods and pharmaceutical compositions of this invention
employ one or more furan nitrones as the active agent. For the purposes of
this
invention, the furan nitrone compounds of formula I are named using
conventional nitrone nomenclature, i.e., the carbon atom of the carbon-
nitrogen
double bond (C=N) is designated the a-position and substituents on the
nitrogen
atom of the carbon-nitrogen double bond are given the N- prefix. For example,
N cyclohexyl-a-(2-sulfofuran-5-yl)nitrone has the formula:
O
iN
H O-O ~ ~ I +
I I
O H
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In some cases, the furan nitrones of this invention may contain one or
more chiral centers. Typically, such compounds will be prepared as a racemic
mixture. If desired, however, such compounds can be prepared or isolated as
pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as
S stereoisomer-enriched mixtures. All such stereoisomers (and enriched
mixtures)
of the furan nitrones of formula I are included within the scope of this
invention.
Pure stereoisomers (or enriched mixtures) may be prepared using, for example,
optically active starting materials or stereoselective reagents well known in
the
art. Alternatively, racemic mixtures of such compounds can be separated using,
for example, chiral column chromatography, chiral resolving agents and the
like.
Additionally, all geometric isomers of the nitrone compounds of formula I
are included within the scope of this invention including, for example, all
isomers (i.e., E and Z isomers) of the carbon-nitrogen double bond of the
nitrone
functionality.
Definitions
When describing the furan nitrones, pharmaceutical compositions and
methods of this invention, the following terms have the following meanings
unless otherwise specified.
"Alkoxy" refers to "alkyl-O-" groups preferably having from 1 to 12
carbon atoms in the alkyl group, more preferably, 1 to 8 carbon atoms.
Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-
propoxy, isopropoxy, n-butoxy, tent-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,
1,2-dimethylbutoxy, and the like.
"Alkoxycarbonyl" refers to the group "-C(O)OR" where R is alkyl.
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"Alkyl" refers to monovalent alkyl groups preferably having from 1 to
about 12 carbon atoms, more preferably 1 to 8 carbon atoms and still more
preferably 1 to 6 carbon atoms. This term is exemplified by groups such as
methyl, ethyl. n-propyl, isopropyl, n-butyl, isobutyl, tent-butyl, n-hexyl, n-
octyl,
tert-octyl and the like. The term "lower alkyl" refers to alkyl groups having
1 to
6 carbon atoms.
"Alkylene" refers to divalent alkylene groups preferably having from 1 to
12 carbon atoms and more preferably 1 to 6 carbon atoms which can be straight
chain or branched. This term is exemplified by groups such as methylene
(-CHZ-), ethylene (-CHZCHZ-), the propylene isomers (e.g., -CHZCHZCHz- and
-CH(CH3)CH,-) and the like.
"Aminocarbonyl" refers to the group "-C(O)NRR" where each R is
independently hydrogen or alkyl, aralkyl or cycloalkyl.
"Aralkyl" refers to "aryl-alkylene-" groups preferably having from 1 to
10 carbon atoms in the alkylene moiety and from 6 to 14 carbon atoms in the
aryl
moiety. Such aralkyl groups are exemplified by benzyl, phenethyl, and the
like.
"Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to
14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed
rings
(e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the
like. Unless otherwise indicated, such aryl groups can optionally be
substituted
with from 1 to 5 substituents, preferably 1 to 3 substituents, selected from
the
group consisting of alkyl, alkoxy, alkoxycarbonyl, carboxyl, cyano, halo,
hydroxy, vitro, thioalkoxy and the like.
"Carboxyl" refers to the group "-C(O)OH" and salts thereof.
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"Cyano" refers to the group "-CN".
"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms
having a single cyclic ring or multiple condensed rings which can be
optionally
substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by
way
of example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl,
cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and
the like, or multiple ring structures such as adamantanyl, and the like.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
"Hydroxy" refers to the group "-OH".
"Nitro" refers to the group "-NOZ".
"Sulfonate" or "sulfo" refers to the group "-S03H" and salts thereof.
"Thioalkoxy" or "alkylthioether" refers to "alkyl-S-" groups. Preferred
thioalkoxy groups include, by way of example, thiomethoxy, thioethoxy, n-
thiopropoxy, isothiopropoxy, n-thiobutoxy and the like.
"Pharmaceutically acceptable salt" refers to salts which are acceptable for
administration to mammals including, by way of illustration, alkali and
alkaline
earth metal salts and addition salts of free acids and amines. Such
pharmaceutically acceptable salts may be derived from a variety of organic and
inorganic counter-ions well known in the art and include, by way of example
only,
sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and
the like; and when the molecule contains a basic functionality, salts of
organic or
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inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate,
acetate,
maleate, oxalate and the like.
The term "pharmaceutically acceptable cation" refers to a pharmaceutically
acceptable cationic counterion of an acidic functional group. Such cations are
exemplified by sodium, potassium, calcium, magnesium, ammonium,
tetraalkylammonium cations, and the like.
n ral vnt_h_etic Procedures
The furan nitrone compounds of this invention can be prepared from
readily available starting materials using the following general methods and
procedures. It will be appreciated that where typical or preferred process
conditions (i.e., reaction temperatures, times, mole ratios of reactants,
solvents,
pressures, etc.) are given, other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the particular
reactants or solvent used, but such conditions can be determined by one
skilled in
the art by routine optimization procedures.
Additionally, as will be apparent to those skilled in the art, conventional
protecting groups may be necessary to prevent certain functional groups from
undergoing undesired reactions. The choice of a suitable protecting group for
a
particular functional group as well as suitable conditions for protecting and
deprotecting various functional groups are well known in the art. For example,
numerous protecting groups, and their introduction and removal, are described
in
T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second
Edition, Wiley, New York, 1991, and references cited therein.
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In a preferred method of synthesis, the furan nitrone compounds of this
invention are prepared by coupling a 5-formylfuran-2-sulfonic acid derivative
of
formula II:
YO-O / \ O II
Ii O.
O
H
wherein Y is as defined above, with a hydroxylamine of formula III:
HO-NH-R' III
wherein Rl is as defined above, under conventional reaction conditions.
The coupling reaction is typically conducted by contacting the sulfonated
furan carboxaldehyde II with at least one equivalent, preferably about 1.1 to
about 2 equivalents, of hydroxylamine III in an inert polar solvent such as
methanol, ethanol, 1,4-dioxane, tetrahydrofuran, dimethylsulfoxide,
dimethylformamide and the like. This reaction is preferably conducted at a
temperature of from about 0°C to about 100°C for about 1 to
about 48 hours.
Optionally, a catalytic amount of an acid, such as acetic acid, hydrochloric
acid,
p-toluenesulfonic acid and the like, may be employed in this reaction.
Additionally, when conducting the coupling reaction, the sulfonate group
is preferably converted into a suitable salt, such as the lithium, sodium or
potassium salt, prior to contacting the hydroxylamine with the sulfonated
furan
carboxaldehyde compound. The sulfonate group is readily converted into the
corresponding salt by contacting the sulfonate with at least one equivalent of
a
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suitable base, such as lithium hydroxide, sodium hydroxide, potassium
hydroxide, sodium hydride and the like.
Upon completion of the coupling reaction, the furan nitrone of formula I
is recovered by conventional methods including precipitation, chromatography,
filtration, distillation and the like.
5-Formyl-2-furansulfonic acid employed in the coupling reaction is
commercially available as the sodium salt hydrate from Aldrich Chemical
Company, Milwaukee, WI 53233. Alternatively, this material can be prepared
from commercially available starting materials using conventional procedures
and reagents.
The hydroxylamine compounds of formula III above are also known
compounds or compounds which can be prepared from known compounds by
conventional procedures. Typically, the hydroxylamine compounds of formula
III are prepared by reducing the corresponding nitro compound (i.e.,
R'-NO2, wherein R' is as defined above) using a suitable catalyst such as an
activated zinc/acetic acid catalyst, activated zinc/ammonium chloride or an
aluminum/mercury amalgam catalyst. This reaction is typically conducted at a
temperature ranging from about 15°C to about 100°C for about 0.5
to 12 hours,
preferably about 2 to 6 hours, in an aqueous reaction media, such as an
alcohol/water mixture in the case of the zinc catalyst or an ether/water
mixture in
the case of the aluminum amalgam catalyst. Aliphatic nitro compounds (in the
form of their salts) can also be reduced to hydroxylamines using borane in
tetrahydrofuran.
Since some hydroxylamines have limited stability, such compounds are
generally prepared immediately prior to reaction with the sulfonated furan
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carboxaldehyde II. Alternatively, hydroxylamines can often be stored (or
purchased commercially) as their hydrochloride salts. In such cases, the free
hydroxylamine is typically generated immediately prior to reaction with the
furan
carbonyl compound by reaction of the hydrochloride salt with a suitable base,
such as sodium hydroxide, sodium methoxide and the like.
Preferred hydroxylamines for use in this invention include, but are not
limited to" N n-butylhydroxylamine, N tent-butylhydroxylamine, N n-
hexylhydroxylamine, N cyclohexylhydroxylamine, N adamantylhydroxylamine
and the like.
Pharmaceutical Compositions
When used as pharmaceuticals, the furan nitrones employed in this
invention are typically administered in the form of a pharmaceutical
composition:
Such compositions can be prepared using procedures well known in the
pharmaceutical art and comprise at least one active compound.
Generally, the compounds of this invention are administered in a
pharmaceutically effective amount. The amount of the compound actually
administered will typically be determined by a physician, in the light of the
relevant circumstances, including the condition to be treated, the chosen
route of
administration, the actual compound administered, the age, weight, and
response
of the individual patient, the severity of the patient's symptoms, and the
like.
The furan nitrone compounds) is typically formulated into a
pharmaceutical composition suitable for oral, parenteral (e.g. intravenous or
intramuscular injection), or rectal (e.g. suppository) administration.
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The compositions for oral administration can take the form of liquid
solutions or suspensions, powders, tablets, capsules or the like. In such
compositions, the furan nitrone of formula I is usually a minor component (0.1
to
about 50% by weight) with the remainder being various vehicles or carriers and
processing aids helpful for forming the desired dosing form. A liquid form may
include a suitable aqueous or nonaqueous vehicle with buffers, suspending
dispensing agents, colorants, flavors and the like.
A solid form may include, for example, any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline cellulose,
gum tragacanth or gelatin; an excipient such as starch or lactose; a
disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening
agent such as sucrose or saccharin; or a flavoring agent such as peppermint,
sugar, methyl salicylate, or orange flavoring.
Injectable compositions are commonly based upon injectable sterile saline
or phosphate-buffered saline or other injectable carriers known in the art.
Again,
the active furan nitrone is typically a minor component, often being from
about
0.05 to 10% by weight, with the remainder being the injectable carrier and the
like.
Rectal administration is usually by suppository. Suppositories are
generally made with a base component of cocoa butter, glycerinated gelatin,
hydrogenated vegetable oils, mixtures of polyethylene glycols of various
molecular weights, or fatty acid esters of polyethylene glycol. The active
furan
nitrone is usually a minor component, often from about 0.05 to 20 % by weight,
with the remainder being the base component.
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The components for orally administrable, injectable compositions and
suppositories are merely representative. Other materials as well as processing
techniques and the like are set forth in Part 8 of ~temineton's Pharmaceutical
i n~s, 18th edition, 1990, Mack Publishing Company, Easton, Pennsylvania,
18042, which is incorporated herein by reference.
One can also administer the compounds of the invention in sustained
release forms or from sustained release drug delivery systems. A description
of
representative sustained release materials can be found in the incorporated
materials in 12P~'>inotnn'~ Pharmaceutical Sciences.
('ondition~ Treated and Treatment Regimens
The conditions treated with the furan nitrone-containing pharmaceutical
compositions of this invention generally include IBD and the various symptoms
which fall within a definition of IBD. The furan nitrone-containing
formulations
are administered to achieve a therapeutic effect. For those furan nitrone
compounds that exhibit a long residency in the body, a once-a-day regimen is
possible. Alternatively, multiple doses, such as up to three doses per day,
typically, may offer more effective therapy. Thus, a single dose or a
multidose
regimen may be used.
In any event, the furan nitrone-containing pharmaceutical composition is
administered in such a manner so that compound is delivered into the patient's
bloodstream. One excellent mode for accomplishing this is intravenous
administration. Intravenous dose levels for treating IBD range from about 0.01
mg/kg/hour of active furan nitrone to about 100 mg/kg/hour, all for from about
1
to about 120 hours and especially 1 to 96 hours. A preloading bolus of from
about 50 to about 5000 mg may also be administered to achieve adequate steady
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state levels. Other forms of parenteral administration, such as intramuscular
injection can be used, as well. In this case, similar dose levels are
employed.
With oral dosing, one to three oral doses per day, each from about 0.1 to
about 150 mg/kg of active furan nitrone are employed, with preferred doses
being from about 0.15 to about 100 mg/kg.
With rectal dosing, one to three rectal doses per day, each from about 1
to about 150 mg/kg of active furan nitrone are employed, with preferred doses
being from about 1 to about 100 mg/kg.
In any treatment regimen, the health care professional should assess the
patient's condition and determine whether or not the patient would benefit
from
furan nitrone treatment. Some degree of routine dose optimization may be
required to determine an optimal doing level and pattern.
A positive dose-response relationship has been observed. As such and
bearing in mind the severity of the side effects and the advantages of
providing
maximum possible amelioration of symptoms, it may be desired in some settings
to administer large amounts of furan nitrone, such as those described above.
The following formulation examples illustrate representative
pharmaceutical compositions of this invention. The present invention, however,
is
not limited to the following pharmaceutical compositions.
Formulation 1 - Tablets
A compound of formula I is admixed as a dry powder with a dry gelatin
binder in an approximate 1:2 weight ratio. A minor amount of magnesium
stearate
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is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90
mg
of active nitrone compound per tablet) in a tablet press.
Formulation 2 - Ca sn ules
A compound of formula I is admixed as a dry powder with a starch diluent
in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules
(125 mg of active nitrone compound per capsule).
Formulation 3 - Liauid
A compound of formula I (125 mg), sucrose (1.75 g) and xanthan gum (4
mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with
a
previously made solution of microcrystalline cellulose and sodium
carboxymethyl
cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color
are
diluted with water and added with stirring. Sufficient water is then added to
produce a total volume of 5 mL.
Formulation 4 - Injection
The compound of formula I is dissolved in a buffered sterile saline
injectable aqueous medium to a concentration of approximately 5 mg/mL.
The following synthetic and biological examples are offered to illustrate
this invention and are not to be construed in any way as limiting the scope of
this
invention.
EXAMPLES
In the examples below, all temperatures are in degrees Celsius
(unless otherwise indicated). Example A and B describe the synthesis of
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intermediates useful for preparing nitrones of this invention; Examples 1-5
describe the synthesis of various nitrones; and the Bioassay Examples describe
the testing of such compounds.
In the examples
below, the following
abbreviations
have the following
meanings. Abbreviations
not defined
below have their
generally accepted
meaning.
bd - broad doublet
bs - broad singlet
d - doublet
dd - doublet of doublets
dec - decomposed
dH20 - distilled water
EtOAc - ethyl acetate
EtOH - ethanol
g - grams
h - hours
Hz - hertz
L - liter
m - multiplet
min - minutes
M - molar
MeOH - methanol
mg - milligram
MHz - megahertz
mL - milliliter
mmol - millimole
m.p. - melting point
N - normal
q - quartet
quint. - quintet
s - singlet
t - triplet
THF - tetrahydrofuran
tlc - thin layer chromatography
~,g - microgram
~,L - microliter
UV - ultraviolet
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Example A
Synthesis of
N tert-Butylhydroxylamine
Zinc dust (648 g) was added in portions to a cooled mixture of 2-methyl-2-
nitropropane (~03 g) and ammonium chloride (207 g) in deionized water (6 L) at
such a rate so as to maintain the temperature below 18°C. The reaction
mixture
was stirred mechanically for 1 S hours and then filtered. The solid was washed
with hot water ( 1.75 L). The combined filtrate was saturated with potassium
carbonate (4.b Kg) and extracted with ethyl acetate (2 x 1300 mL). The organic
solution was dried over anhydrous sodium sulfate, filtered and rotary
evaporated to
give the title compound (329 g, 75.7% yield) as white crystals. This material
was
used without further purification.
Spectroscopic data were as follows:
'H NMR (CDCl3, 270 MHz) 8 = 1.090 (s, 3 CH3).
Example B
Synthesis of
N Cyclohexylhydroxylamine
Using the procedure of Example A above and nitrocyclohexane, the title
compound can be prepared. Alternatively, N-cyclohexylhydroxylamine
hydrochloride may be purchased commercially from Aldrich Chemical Company,
Inc., Milwaukee, WI USA and neutralized with a base, such as potassium
carbonate, to provide the title compound.
Example 1
Synthesis of
N n-Butyl-a-(2-sulfofuran-5-yl)nitrone
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N n-Butylhydroxylamine (0.1 mol) and 5-formylfuran-2-sulfonic acid,
sodium salt hydrate (0.1 mol) were refluxed in methanol (200 mL) for 24 hours.
Another portion of N n-butylhydroxylamine was added and the reaction stirred
for 24 hours. The solvent was stripped to provide a pale yellow solid which
was
recrystallized from ethyl acetate to afford the title compound in 6.7 % yield
as the
sodium salt, m.p. 212.8°C (dec).
Spectroscopic data was as follows:
IR (KBr, cm'): 2959.2 (CH), 2931.9 (CH), 1636 (C=N), 1246 (SO3),
1225.2 (S03) and 1165.4 (N-O).
'H NMR (DMSO-d6, 270 MHz): 8 = 8.139 (1H, s, nitronyl CH), 7.677
(1H, d, J = 3.7 Hz, furan CH), 7.047 (1H, d, J = 3.7 Hz, furan CH), 4.026
(2H, t, J = 6.9 Hz, CH2), 1.889 (2H, m, CHZ), 1.373 (2H, m, CHZ) and 0.950
(3H, t, J = 7.4 Hz, CH3).
'3C NMR (DMSO-d6, 270 MHz): 8 = 153.699, 146.774, 130.713,
118.037, 113.766, 64.742, 28.561, 18.540 and 12.438.
Example 2
Synthesis of
N tent Butyl-a-(2-sulfofuran-5-yl)nitrone
Following the procedure of Example 1 above and using 5-formylfuran-2-
sulfonic acid, sodium salt hydrate and N tent-butylhydroxylamine, the title
compound was prepared in 36% yield as the sodium salt, m.p. 117-120°C
(dec.).
Spectroscopic data was as follows:
'H NMR (DMSO-d6, 90 MHz): 8 = 7.908 (1H, s, nitronyl CH), 7.445
(1H, d, J = 3.3 Hz, furan CH), 6.486 (1H, d, J = 3.3 Hz, furan CH), and
1.430 (9H, s. 3 CH3).
Example 3
Synthesis of
N n-Hexyl-a-(2-sulfofuran-5-yl)nitrone
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Following the procedure of Example 1 above and using 5-formylfuran-2-
sulfonic acid, sodium salt hydrate and N n-hexylhydroxylamine, the title
compound was prepared in 76% yield as the sodium salt, m.p. 225.5°C
(dec.).
Spectroscopic data was as follows:
IR (KBr, cni'): 2956.8 (CH), 2927.1 (CH), 1617.2 (C=N), 1247.6
(503), 1222.7 (503) and 1171.2 (N-O).
'H NMR (DMSO-db, 270 MHz): 8 = 8.050 (1H, s, nitronyl CH), 7.448
(1H, d, J = 2.2 Hz, furan CH), 6.517 (1H, d, J = 2.2 Hz, furan CH), 3.890
(2H, t, J = 6.6 Hz, CHZ), 1.779 (2H, m, CHZ), 1.263 (2H, m, CHZCHZCHZ) and
0.850 (3H, t, CH3).
'3C NMR (DMSO-db, 270 MHz): 8 = 158.275, 146.866, 125.069,
113.964, 110.258, 64.772, 30.895, 27.142, 25.526, 22.078 and 13.948.
Example 4
Synthesis of
N Cyclohexyl-a-(2-sulfofuran-5-yl)nitrone
Following the procedure of Example 1 above and using 5-formylfuran-2-
sulfonic acid, sodium salt hydrate and N cyclohexylhydroxylamine, the title
compound was prepared in 84.3% yield as the sodium salt, m.p. 236.1°C
(dec.).
Spectroscopic data was as follows:
IR (KBr, ctri'): 2934.2 (CH), 2858.4 (CH), 1637.2 (C=N), 1215.5 (503)
and 1168.9 (N-O).
'H NMR (DMSO-d6, 270 MHz): 8 = 8.015 (1H, s, nitronyl CH), 7.439
(1H, d, J = 3.5 Hz, furan CH), 6.508 (1H, d, J = 3.5 Hz, furan CH), 4.053
(1H, m, NCH) and 1.814-1.097 (IOH, m, 5 CHZ).
'3C NMR (DMSO-db, 270 MHz): 8 = 158.230, 147.049, 123.208,
113.858, 110.197, 72.597, 30.666, 24.839 and 24.534.
Example 5
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Synthesis of
N Adamantyl-a-(2-sulfofuran-S-yl)nitrone
Following the procedure of Example 1 above and using 5-formylfuran-2-
sulfonic acid, sodium salt hydrate and N adamantylhydroxylamine, the title
compound was prepared in 59% yield as the sodium salt, m.p. 236.9°C
(dec.).
Spectroscopic data was as follows:
IR (KBr, cm'): 2910.7 (CH), 2853.0 (CH), 1638 (C=N), 1216.8 (503)
and 1168.4 (N-O).
'H NMR (DMSO-d6, 270 MHz): 8 = 7.849 (1H, s, nitronyl CH), 7.495
(1H, d, J = 3.5 Hz, furan CH), 6.518 (1H, d, J = 3.5 Hz, furan CH), 2.166
(3H, bs, 3 CH), 2.075 (6H, bs, 3 CHZ) and 1.666 (6H, bs, 3 CH2).
'3C NMR (DMSO-d6, 270 MHz): b = 158.306, 147.644, 120.417,
114.087, 110.288, 69.669, 35.639 and 29.293.
Bioassay Example 1
Evaluation of Compounds in TNBS Model for IBD
In this experiment, the ability of compounds of this invention to reduce
colonic inflammation is demonstrated using the trinitrobenzene sulphonic acid
("TNBS") model for IBD. The TNBS model is one of the standard IBD models
used in IBD discovery research and it has been extensively evaluated in
rodents.
See, for example, C. O. Elson et al. (1995), Experimental Models of
Inflammatory Bowel Disease, Gastroenterology, 109: 1344-1367 and references
cited therein. In this model, a single enema of TNBS induces a prolonged
colonic inflammatory response (up to several weeks) that is transmural and is
accompanied by oxidative damage as evidenced by an increase in
myeloperoxidase ("MPO") activity. Additionally, the inflammation is
characterized by discrete areas of acute necrosis, inflammation and muscle
thickening. Agents with anti-inflammatory effects in patients with IBD show
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efficacy in this model. Although the mechanism by which TNBS induces an
inflammatory response is unknown, it is thought to have an immunological
basis.
Male Sprague-Dawley rats (200-250 g) were housed in standard cages (2
per cage) and fed rat chow and tap water ad libitum. After an overnight fast,
rats
were brought into the laboratory and randomized into treatment groups. Colitis
was induced by intrarectal administration of 0.5 ml of TNBS solution (50 mg/kg
in 50% ethanol) using a 1 mL syringe attached to a 5 cm polyethylene catheter.
Control animals received saline (0.9 % ) or a 1 % methyl cellulose suspension
at
identical time points.
Tissue Analvsis
Three days after TNBS administration, the rats were sacrificed and the
colons excised and opened longitudinally. In 5 cm segments of colon, gross
morphology was determined using the following scale:
~ 11 TindinQ
_.-,
0 No damage
1 One area of Inflammation (red),
no ulcers
2 Ulcers, no area of inflammation
3 Ulcers, dne area of inflammation
4 More than 2 ulcers, inflammation
at one site
5 More than 2 ulcers inflammation
> 1 cm
The weights of each 5 cm colonic segment were also recorded to assess
inflammatory induced edema.
Dosing Regimen
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Each of the compounds from Examples 1-9 were tested in the TNBS
model at 10 mg/kg p.o. (oral) dosing. Each of the test compounds was
administered by oral gavage as a 1 % carboxy methyl cellulose ("CMC")
suspension 1 hour prior to the administration of TNBS. Control rats were given
CMC only .
Each of the test compounds reduced TNBS-induced damage compared to
the controls. The reduction in TNBS-induced damage ranged from about 25 % to
about 44 % .
Bioassay Example 2
Mouse Dextran Sulfate IBD Model
Another model used for screening candidate IBD-treating compounds is
the Dextran Sulfate ("DSS") model. Similar to the TNBS model, DSS induced
colitis is widely used as a screening tool for IBD therapeutics. When
administered orally, DSS induces IBD-like symptoms in Swiss-Webster mice.
This model can be used to determine the effectiveness of compounds of this
invention when such compounds are administered orally (p.o.).
Individually housed 30-40 g male Swiss-Webster mice (B & K Universal,
Fremont, CA) receive 3 % DSS (Sigma Chemicals, St. Louis, MO) in their
drinking water for 7 days. All animals receive food and water ad libitum.
Two groups of mice are dosed orally with either the test compound in a
dosing vehicle (1 % methyl cellulose, dose range of 10 mg/kg to 30 mg/kg) or
dosing vehicle alone (control).
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Clinical signs of colitis are assessed by a disease activity index ("DAI")
consisting of changes in stool characteristics, fecal occult bleeding and body
weight loss. The DAI is very similar to the Crohn's Disease Activity Index
used
in clinical trials to evaluate new agents to prevent/treat IBD. The DAI data
are
analyzed using Proc Anova in SAS with a Bonferoni post-hoc analysis, and
Model 108 in WinNonlinT'" (Professional Version 1.5, Scientific Consulting,
Apex, NC) for the EDSO and EmaX values. The wet weight and myeloperoxidase
("MPO") data (collected only on Day 7) are analyzed by Proc TTest in SAS.
MPO is a marker for neutrophil infiltration. The following criteria are
employed
in this assay:
DAI Scoring_,(Dailv)
Stool Characteristic: 0 = normal, 2 = loose and 4 = diarrhea
Fecal Occult Blood: 0 = negative, 2 = positive, 4 = gross bleeding
Weight Change: 0 = 0-1 % , 1 = 1 to < 5 % , 2 = 5 % to < 10 % , 3 = 10
to < 20 % , 4 = > 20
MPO (pay 7 Onlvl
Two strips of colonic tissue/mouse
MPO activity by spectrophotometric assay
Bioassay Example 3
Establishment of the Dose-response Characteristics in the Mouse Dextran
Sulfate Model
To determine the dose-response relationship of a test compound in the
DSS Mouse Model, the following procedure is used.
Experimental conditions and statistical analyses are the same as the
Mouse Dextran Sulfate IBD Model, except four groups of mice (n = 8-l0/group)
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are used. Animals are dosed orally with either test compound (3, 10 or 30
mg/kg) or vehicle alone. In addition, the following procedure is introduced to
evaluate the histology in the animals:
5-6 slices/segment with 15-18 total pieces/colon
Score for extent of damage: 0 = 1-25 % involvement, 1 = 26-50%
involvement, 2 = 51-75 % involvement, 3 = 76-100% involvement
Score for grade:
0 = intact crypt, 1 = loss of 1/3 crypt, 2 = loss of 213 crypt, 3
= loss of entire crypt with surface epithelium intact, 4 = loss of
entire crypt and erosion of surface epithelium
Score for Severity:
0 = normal, 1 = focal inflammatory cell infiltrate including
PMNs, 2 = inflammatory cell infiltration, gland dropout and
crypt abscess, 3 = mucosal ulceration
Single, evaluator (qualified pathologist) blinded to the treatment
conditions.
Bioassay Example 4
Effect of Test Compounds on Flux of Reactive Oxygen Species Induced by
TNF-a
Oxidative stress agents (OSA) are thought to be involved in cell death in
1BD and are key initiator in the cascade of events leading to apoptosis. The
purpose of this study is to evaluate the effect of a test compound on cytokine-
induced OSA flux.
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To visualize OSA, the dye dihydrodichlorofluorescein diacetate is used.
This non-fluorescent dye is taken up by cells and deacetylated to its non-
fluorescent congener dihydrodichlorofluorescein (HZDCF), which is trapped
within cells. Reactive oxygen species ("ROS") react with HZDCF, converting it
to the highly fluorescent DCF. DCF fluorescence can be measured
spectrofluorometrically and can also be visualized in intact cells using
fluorescent
microscopy.
SK-N-MC cells (American Type Culture Collection, Rockville, MD) are
plated at 250,000 cells/well in 24-well Corning plates. Following plating, the
cells are maintained in retinoic acid medium (5 ~,M) for five days and then
treated with a test compound at 100~,M for 1 hour prior to TNF-a (3.0 ng/mL)
treatment. TNF-a and HZDCF are added simultaneously and cultures are
incubated for an additional 4 hours. Following incubation, cultures are read
in a
cytofluorometer at 485-530 nm wavelength to detect increased DCF formation.
Relative fluorescence units (RFU) values for the respective treatment
conditions
are compared. In this assay, higher fluorescence readings indicate ROS
production. Thus, reductions in fluorescence indicates reduction in ROS
production.
Bioassay Example 5
Effect of Compound A on TNF-a Induced Apoptosis in a Human Cell Model
This test is used to evaluate the potential of a test compound to prevent
TNF-a induced apoptosis.
A test compound is evaluated in an in vitro model of TNF-a induced
toxicity (see Pulliam et al. J. Neurosci. Res. 21:521-530 (1998)). In this
model,
human brain cell aggregates from fetal tissue are treated with TNF-a which
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caused an apoptotic cell death. Brain cell aggregates prepared from 1 brain
were
incubated for 10-12 days before experimentation. Aggregates are weighed out
(100 mg/flask) and aliquoted into 10 mL flasks. TNF-a is used at a
concentration
of 1 ng. The test compound is added 1 hour prior to the TNF-a. Experiments
include untreated brain aggregates, TNF-a-treated brain aggregates, TNF-a- +
test compound treated aggregates and test compound treated aggregates. After
TNF-a is added, aggregates are incubated for an additional 48 h. After this
time, brain aggregates are centrifuged for 5 min at 500 rpm. The supernatant
is
removed and the pellet is lysed for determination of programmed cell death
(Boeringer Mannheim Cell Death Kit ELISA).
Bioassay Example 6
Effect of Test Compound on TNF-a Induced Reduction in bcl-2
Cytokine-mediated apoptosis or programmed cell death is believed to be
involved in a number of diseases including IBD. Reductions in bcl-2 are a
major
signal in initiation of the apoptotic cascade (see Jourd'heuil et al., J. Clin
Gastroenterol. 25(Suppl):561-S72 (1997)). The purpose of this study is to
investigate the effects of a test compound on bcl-2 protein levels in a
cellular
model of cytokine mediated apoptosis.
SK-N-MC cells (American Type Culture Collection, Rockville, MD) are
plated at 500,000 cells/plate and treated with retinoic acid ("RA") (5 ~,M)
for 5
days. Following RA treatment, the cells are incubated with a test compound
(100 ~,M) for 1 hour. Cells are then treated with increasing concentrations of
TNF-a (0, 0.3 and 3 ng/mL) for 6 h. The cells are harvested and lysed and bcl-
2 is measured in the lysate using an ELISA assay (Boehringer Manheim).
Quantification of bcl-2 is based on a standard curve and results are expressed
as
units/mL of bcl-2 in the sample.
